In the intestine, effective epithelial barrier formation requires the presence of tight junctions, which regulate the passage of ions and molecules through the paracellular space. Defects associated with the tight junction contribute to barrier dysfunction, diarrhea, and extensive nutrient and protein loss in intestinal diseases. In immune-mediated intestinal diseases, cytokines secreted in the lamina propria are thought to be a primary driver of barrier loss. My preliminary in vitro studies demonstrate that two cytokines known to be elevated in inflammatory bowel disease, IL-13 and TNF, induce barrier dysfunction through functionally distinct pore and leak pathways. IL-13 induces the formation of tight junction claudin-2 pores resulting in increased permeability of small cations, but not large ions o macromolecules. In contrast, TNF increases non-selective tight junction leak to small and large ions and molecules by inducing occludin internalization. Further, I have shown that these two pathways are differentially regulated by casein kinase II dependent regulation of claudin- 2 pore stability and MLCK dependent internalization of occludin. During my K08 studies, I have developed an approach to study local tight junction function in submicron segments of tight junction. This has permitted me to define tight junction function with unprecedented molecular resolution. While my K08 award continues to support my ongoing studies of how these pathways are regulated in cultured epithelial monolayers, these R03 studies will define regulation of pore and leak pathway function in a more complex in vivo setting. My central hypothesis is that the relative contribution of pore and leak pathway permeability depends on the interplay of different classes of dynamic tight junction channel openings and closings with varying size and charge selectivity properties. To test this hypothesis in vivo, I intend to study regulation of pore and leak pathways in intact mouse mucosa. Using global measurements I will determine the mechanisms that regulate tight junction claudin-2 pore function as well as occludin- and tricellulin-dependent leak permeability. In addition, I will adapt my local patch clamp approach to study the role of claudin-2, occludin, and tricellulin to local tight junction opening events in mouse mucosa. In summary, these studies will define the relative contribution and molecular aspects of pore and leak pathway function in vivo. The results are expected to be directly applicable to understanding the roles of TNF and IL-13 in intestinal diseases such as IBD, and are further expected to provide novel approaches to study barrier dysfunction in patients in the near future. This will aid in the rational development of specific therapies targeting molecular alterations responsible for barrier dysfunction.